I. Methods for the Identification of Promoters
Many systems have been used to isolate genes and their promoters located immediately upstream of the translation start site of a gene. The techniques can roughly be divided in two categories, namely (1) where the aim is to isolate genomic DNA fragments containing promoter activity randomly by so-called promoter probe vector systems and (2) where the aim is to isolate a gene per se from a genomic bank (library) and isolation of the corresponding promoter follows therefrom.
In promoter probe vector systems, genomic DNA fragments are randomly cloned in front of the coding sequence of a reporter gene that is expressed only when the cloned fragment contains promoter activity (Neve, R. L. et al., Nature 277:324-325 (1979)). Promoter probe vectors have been designed for cloning of promoters in E. coli (An, G. et al., J. Bact. 140:400-407 (1979)) and other bacterial hosts (Band, L. et al., Gene 26:313-315 (1983); Achen, M. G., Gene 45:45-49 (1986)), yeast (Goodey, A. R. et al., Mol. Gen. Genet. 204:505-511 (1986)) and mammalian cells (Pater, M. M. et al., J. Mol. App. Gen. 2:363-371 (1984)). Because it is well known in the art that Trichoderma promoters fail to work in E. coli and yeast (e.g. Penttila, M. E. et al., Mol. Gen. Genet. 194:494-499 (1984)), these organisms cannot be used as hosts to isolate Trichoderma promoters. Due to the fact that, during the transformation of Trichoderma, the transforming DNA integrates into the fungal genome in varying copies in random locations, application of this method by using Trichoderma itself as a cloning host is also unlikely to succeed and would not be practical for efficient isolation of Trichoderma promoters with the desired properties.
Known genes can be isolated from either a cDNA or chromosomal gene bank (library) using hybridization as a detection method. Such hybridization may be with a corresponding, homologous gene from another organism (e.g. Vanhanen et al., Curr. Genet. 15:181-186 (1989)) or with a probe designed on the basis of expected similarities in amino acid sequence. If amino acid sequence is available for the corresponding protein, an oligonucleotide can also be designed which can be used in hybridization for isolation of the gene. If the gene is cloned into an expression bank, the expression product of gene can be also detected from such expression bank by using specific antibodies or an activity test.
Specific genes can be isolated by using complementation of mutations in E. coli or yeast (e.g. Keesey, J. K. et al., J. Bact. 152:954-958 (1982); Kaslow, D. C., J. Biol. Chem. 265:12337-12341 (1990); Kronstad, J. W., Gene 79:97-106 (1989)), or complementation of corresponding mutants of filamentous fungi for instance by using SIB selection (Akins et al., Mol. Cell. Biol. 5:2272-2278 (1985)).
However, a major concern is how to isolate specific genes that have the desired promoter properties, for example genes which would be most highly expressed when glucose is present in the medium. There is no information available in the literature to indicate which genes are the most highly expressed in an organism, and especially not from filamentous fungi. The phosphoglyceratekinase (PGK) promoter from the yeast Saccharomyces cerevisiae is considered to be a strong promoter for protein production. However, results obtained by the inventors have shown that the corresponding Trichoderma promoter is not suitable for such protein production. Thus, the identification of specific Trichoderma genes for their isolation in order to obtain the best possible promoter for protein production in certain desired conditions is unknown and cannot be predicted. Consequently one cannot rely on any previous nucleotide or amino acid sequence information, nor complement any previously known mutations, in gene isolation for such purpose in Trichoderma.
Differential hybridization has been used for cloning of genes expressed under certain conditions. The method relies on the screening of a bank separately with an induced and noninduced cDNA probe. By this method e.g. Trichoderma reesei genes strongly expressed during production of cellulolytic enzymes have been isolated (Teeri, T. et al., Bio/Technology 1:696-699 (1983)). The differential hybridization methods used are based on the idea that the genes searched for are expressed in certain conditions (like cellulases on cellulose) but not in some other conditions (like cellulases on glucose) which enables picking up clones hybridizing with only one of the cDNA probes used. However, for isolation of the genes expressed strongly on glucose, this approach (expression on glucose and not on some other media) is not a suitable one, and might in fact result in not finding the most highly expressed genes. This is because when differentially screening a chromosomal bank, only induced genes are selected. Such induced genes are not necessarily the most strongly expressed genes. Thus, no method is known in the art which would permit the identification of promoters which function strongly in Trichoderma on glucose medium.
Another option for obtaining a promoter with desired properties is to modify the already existing ones. This is based on the fact that the function of a promoter is dependent on the interplay of regulatory proteins which bind to specific, discrete nucleotide sequences in the promoter, termed motifs. Such interplay subsequently affects the general transcription machinery and regulates transcription efficiency. These proteins are positive regulators or negative regulators (repressors), and one protein can have a dual role depending on the context (Johnson, P. F. and McKnight, S. L. Annu. Rev. Biochem. 58:799-839 (1989)). However, even a basic understanding of the regions responsible for regulation of a promoter requires a considerable amount of experimental data, and data obtained from the corresponding promoter of another organism is usually not useful (see Vanhanen, S. et al., Gene 106:129-133 (1991)), or at least not sufficient, to explain the function of a promoter originating from another organism.
II. Translation Elongation Factors
Translation Elongation Factors (TEFs) are universally conserved proteins that promote the GTP-dependent binding of an aminoacyl-tRNA to ribosomal A-site in protein synthesis. Especially conserved is the N-terminus of the protein containing the GTP binding domain. TEFs are known as very abundant proteins in cells comprising about 4-6% of total soluble proteins (Miyajima, I. et al., J. Biochem. 83:453-462 (1978); Thiele, D. et al., J. Biol. Chem. 260:3084-3089 (1985)).
tef genes have been isolated from several organisms. In some of them they constitute a multigene family. Also a number of pseudogenes have been isolated from some organisms. The promoter of the human tef gene can direct transcription in vitro at least 2-fold more effectively than the adenovirus major late promoter, which indicates that the tef promoter is a strong promoter in mammalian expression systems (Uetsuki et al., J. Biol. Chem. 264:5791-5798 (1989)). Both the human and the A. thaliana tef1 promoter (for translation elongation factor EF-1.alpha.) has been used in an expression system with high efficiency of gene expression (Kim et al., Gene 91:217-223 (1990); Curie et al., Nucl. Acid Res. 19:1305-1310 (1991)). In both cases the full expression of the promoter was dependent on the presence of the intron in the 5' noncoding region.
tef is quite constitutively expressed, the major exception being its expression in aging and quiescent cells. It is not known to be regulated by the growth substrates of the host.
III. Expression of Recombinant Proteins in Trichoderma
The filamentous fungus Trichoderma reesei is an efficient producer of hydrolases, especially of different cellulose degrading enzymes. Due to its excellent capacity for protein secretion and developed methods for industrial cultivations, Trichoderma is a powerful host for production of heterologous, recombinant proteins in large scale. The efficient production of both homologous and heterologous proteins in fungi relies on fungal promoters. The promoter of the main cellulase gene of Trichoderma, cellobiohydrolase 1 (cbh1), has been used for production of heterologous proteins in Trichoderma grown on media containing cellulose or its derivatives (Harkki et al., Bio/Technology 7:596-603 (1989); Saloheimo et al., Bio/Technology 9:987-990 (1991)). The cbh1 promoter cannot be used when the Trichoderma are grown on glucose containing media due to glucose repression of cbh1 promoter activity. This regulation occurs at the transcriptional level and thus glucose repression could be mediated through the promoter sequences. However, nothing is yet known of the mechanism of glucose repression at the promoter level in filamentous fungi.
Glucose repression in the yeast Saccharomyces cerevisiae has been studied for many years. These studies have however failed, until recently, to identify binding sequences in promoters or regulatory proteins binding to promoters which would mediate glucose repression. The first ever published glucose repressor protein and the binding sequence in eukaryotic cells was published by Nehlin and Ronne (Nehlin, J. O. and Ronne, H. EMBO J. 9:2891-2899 (1990)). This MIG1 protein seems to be responsible of one fifth of the glucose repression of GAL genes in Saccharomyces cerevisiae, other factors still being required to obtain full glucose repression effect (Nehlin, J. O. et al., EMBO J. 10:3373-3377 (1991)).
Thus, it is desirable to be able to produce proteins in Trichoderma grown on glucose. Not only is the substrate glucose cheap and readily available, but also Trichoderma produces less protease activity when grown on glucose. Further, cellulase production is repressed when Trichoderma is grown on glucose, thus allowing for the easier purification of the desired product from the Trichoderma medium. Nevertheless, to date there has been no identification or characterization of any promoter that is highly functional in Trichoderma grown on glucose. In addition, no modifications of the normally glucose repressed promoter, the cbh1 promoter, have been identified which would allow the use of this strong promoter for expression of heterologous genes in Trichoderma grown on glucose.